Cell-mediated immunity (CMI) imposes a strong selective pressure on HIV evolution (adaptation). Recent research has generated clear evidence that the transmitted/founder viruses (TFV) often contain both adapted and non-adapted (cytotoxic T lymphocyte) CTL epitopes and that immune escape mutations are highly predictable based on host immunogenetic profiles. The central hypothesis of this grant is that effective (durable) immune control of HIV infection is due in large part to a combination of (i) th transmission of a TFV with low viral replicative capacity (vRC), (ii) efficient targeting of conserved, non-adapted CD8 and CD4 T-cell epitopes, and (iii) favorable immunogenetic features, in the newly infected hosts. To gain a better understanding of causal pathways to protective CTL responses, this multi-PI R01 project will continue to focus on studying early immune responses in African sero-converters (SCs) with well-defined viral and host characteristics in a large cohort of acutely and chronically infected subjects with two major HIV subtypes. The new research activities will build upon a solid ground already laid out by ongoing studies of two major infecting viral subtypes (A and C). Specifically, Aim 1 will examine the diversity of TFV initiating infection, through analyses of full-length sequences of TFV (acute infection) and their progenies at 3 and 12 months after estimated date of infection (EDI) in 200 acutely infected individuals (80 subtype A from Rwanda, 120 subtype C from Zambia). Infectious molecular clones (IMCs) representing all 200 TFV will be tested in vitro for viral replicative capacity (vRC), while additional analyses will define relative fitness costs of CTL escape mutations that emerge early (3 months) or later in infection (12 months). Aim 2 will study early CD4 and CD8 T-cell responses that translate to effective immune control in all 200 acutely infected SCs with fully resolved HLA class I and II genotypes. These analyses will focus on the impact of TFV pre-adaptation on CD4 and CD8 responses using novel cohort-specific, potential T-cell epitopes (PTEs). T-cells and T-cell clones activated by autologous epitopes will be tested for in vitro efficacy in viral inhibition. To strengthen research under the first two aims, Aim 3 wll utilize fine mapping techniques on an extended HLA region to validate causal immunogenetic correlates based on an expanded cohort consisting of all available SCs and seroprevalent subjects (SPs). Novel biostatistical methods will allow us to fully integrate this multifactorial approach into a definitive elucidation of viral and host factors that account for virologic and immunologic control. Overall, these studies on HIV virology, immunology, and human genetics will provide critical and valuable insights for improving HIV vaccine design, especially in terms o novel strategies for inducing protective immunity and enhancing vaccine immunogenicity critical to prophylaxis and a functional cure.